Studies on the immunopathogenesis, diagnosis and control of infectious bronchitis and avian metapneumoviruses in chicken

This thesis describes field and experimental investigations on various aspects of the immunopathogenesis, diagnosis and vaccination of infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) in the chicken. The immunopathogenesis of an economically important variant IBV (IS/885/00 like) seen in the Middle East and North Africa was examined in one day old specific pathogen free (SPF) and commercial broiler chicks (Chapter 3). The virus caused respiratory distress, depression and diarrhoea in both SPF and broiler chicks but the severity was milder in the latter. Mild head swelling was observed in one infected broiler chick at 15 days post infection (dpi) and virus with 100% nucleotide level similarity to the inoculum was detected by reverse transcription polymerase chain reaction (RT-PCR) and virus isolation (VI). In the IS/885/00-like infected SPF chicks, cystic oviducts were found in two female chicks. IBV with 99% part S1 sequence similarity to the initial inoculum was isolated from the cystic fluid. The protection provided by current commercial vaccines against variant IBV IS/885/00 like and IS/1494/00 like was investigated in day old commercial broiler chicks (Chapter 4). Protection was evaluated based on the clinical signs, gross lesions, tracheal ciliary scores and virus detection by RT PCR. It was found that administering combined live H120 and CR88 vaccines simultaneously at day old, followed by CR88 vaccine at 14 days old gave more than 80% ciliary protection against both of the Middle East isolates. Cellular and local immune responses in the trachea following vaccination of day old broiler chicks with different live IBV vaccines were evaluated (Chapter 5). In addition, protection conferred against virulent IBV was also examined. All vaccination programmes were able to induce measurable levels of CD4+, CD8+ and IgA bearing B cells in the trachea following vaccination when compared to unvaccinated birds. Expression levels of CD4+ and CD8+ cells varied between the vaccinated groups. Vaccines containing Mass2 combined with 793B2 produced good protection against challenge with virulent IBV QX compared to vaccines containing Mass (Mass1 or Mass2) alone or Mass1 with D274 or CR88. All vaccination programmes produced more than 80% protection against homologous (M41 and 793B) challenge. In Chapter 6, IBVs with high nucleotide level similarity to IS/885/00 like and IS/1494/06 like strains were detected by RT PCR in a broiler flock exhibiting high mortality and respiratory distress in Libya. For the first time, these findings have highlighted the circulation of variant IBVs in the Eastern part of Libya. Humoral and cellular immune responses and protection studies in SPF chicks that received live Newcastle disease virus (NDV), aMPV and IBV vaccines in single, dual or triple combinations were examined (Chapter 7). Protection against virulent IBV or aMPV was not affected when the vaccines were given either singly or in combination. There were no significant differences in the mean antibody titres of the NDV-vaccinated groups and they remained above the protective titre. The mean titres of antibodies against aMPV were suppressed when aMPV vaccine was given with other live vaccines but the aMPV-vaccinated groups were fully protected when challenged with virulent aMPV. The mean titres of antibodies were similar in the IBV-vaccinated groups and all IBV-vaccinated groups gave almost 100% protection against M41 challenge. Between the vaccinated groups, there were no significant differences in the mean numbers of CD4+, CD8+ and IgA-bearing B cells, reflecting similar levels of tracheal cellular and IgA responses irrespective of single, dual or triple vaccine applications. Despite the aMPV humoral antibody suppression, the efficacy of the live vaccines was not compromised when they were given simultaneously to young SPF chicks. Comparative studies in day old SPF chicks using both aMPV subtype A or B, separately or in combination, were evaluated (Chapter 8). There were significant differences in the degree of the clinical signs induced by the single subtypes A, B or A+B given together, with most severe signs observed in the latter two groups. By RT-PCR or VI, subtype B virus persisted longer than subtype A. Even though similar titres of the viruses were used, birds given subtype B alone or in combination showed a greater increase in antibody titres than those given A. These findings demonstrate that for the two strains used, subtype B was more pathogenic than subtype A and was excreted and persisted in the tissues for longer. The use of Flinders Technology Associates (FTA) cards for detection of several avian pathogens has been previously reported. To date, no information has been published on the use of FTA cards for detection of aMPV. In Chapter 9, the feasibility of using FTA cards for the molecular detection of aMPV subtype A and B by RT-PCR was investigated. Findings showed that FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization